Monoclonal antibodies are typically produced by fusing an antibody producing cell with an immortalizing cell to generate a homogenous population of cells all producing antibodies directed to the same antigenic epitope. Mice and rats are traditional sources for the antibody producing cells. Cell hybrids produced from the fusion of the antibody producing cell and the immortal fusion partner, termed fusion products of hybrids, are selected and tested for the quality and quantity of secreted antibodies reacting with the immunizing antigen. The hybrids are expanded in vitro or in vivo, as ascites for large scale antibody production. For a review of current procedures used to develop monoclonal antibodies, see Waldman, T., Science 252:1657-1662 (1991) and Harlow, et al., Antibodies: A Laboratory Manual. Cold Spring Harbor, 1988. New York.
Monoclonal antibodies are preferred over polyclonal antibodies for diagnostic assays. Monoclonal antibodies represent a homogenous population of antibody with a defined specificity. The antibody can be used repeatedly over time with consistent results. Since the monoclonal population is homogenous it tends to have a more predictable reaction pattern in immunoassays over its polyclonal counterpart. Moreover, in contrast to polyclonal antibodies, monoclonal antibodies of consistent quality can be generated over a prolonged period. The specificity of monoclonal antibodies makes them the preferred type of antibody for immunoassays such as enzyme linked immunosorbent assays (ELISA), radioimmunoassays (RIA), western blot or immunohistochemical assays.
While rats and mice are the typical sources for monoclonal antibody production, some antigens do not sufficiently stimulate the development of high-affinity antibodies in mice and rats to make them useful in diagnostic assays. Examples of this type of antigen primarily include carbohydrate moieties. While amino acid epitopes tend to be more immunogenic than carbohydrates, there are amino acid epitopes that do not stimulate high affinity antibodies in rodents. Therefore another convenient source of monoclonal antibodies would be of great benefit to the art.
Rabbits are an excellent source of polyclonal antibodies. Antigen stimulation, in rabbits, using a wide variety of immunogens, consistently generates high titer rabbit polyclonal antibodies. Moreover, rabbits are a common and convenient laboratory animal. While rodents are less likely to produce high-affinity antibody to carbohydrate moieties, rabbits more consistently react positively to foreign carbohydrate determinants. The difference in affinity between rabbits and mice may be attributed to the fact that these organisms are evolutionarily disparate and may therefore respond differently to the same antigen. Thus rabbits may be better able to generate stronger antibody responses to those human epitopes that are weakly reactive in mice. Carbohydrate determinants are important markers for cancer diagnosis and infectious disease; thus there is an increasing need for high affinity antibodies to these antigens.
In addition to the different antigenic responses between rabbit and mice, antibodies produced in rabbits offers additional advantages to diagnostic assays. Some humans, for example, tend to have endogenous levels of anti-murine antibodies (HAMA). In standard ELISA assays that employ two murine antibodies, this antibody cross-links the two murine antibodies to generate false positive signals. Reduced false positives are an important goal in immunoassay development. Therefore mechanisms to reduce false positives in diagnostic assays are important to health care personnel. Endogenous human anti-rabbit antibodies are uncommon. Additionally, human anti-mouse antibodies may complicate therapeutic uses for murine antibodies as well. For example, the ability to detect an antigen associated with cancer in an individual who is imaged or treated with murine monoclonal antibodies to that same antigen may be compromised in a diagnostic assay that employs the murine monoclonal-monoclonal format. A rabbit-rabbit double monoclonal format or a rabbit monoclonal-murine monoclonal format would obviate the need for a mouse-mouse monoclonal format.
While monoclonal antibodies are generally preferred over polyclonal antibodies in immunoassays, there is no efficient method available to produce rabbit monoclonal antibodies in a consistent quantity or quality. There are no rabbit myelomas or suitable immortalizing fusion partners derived from rabbits. Other methods for producing rabbit monoclonal antibodies include B cell transformation in rabbits using SV40 (simian virus 40) or EBV (Epstein Barr Virus) and oncogene transfection. All of these methods have proven difficult and results are inconsistent (see Collins, et al., Proc. Natl. Acad. Sci. 71:260-262, 1974 and Strosberg, et al., U.S. Pat. No. 4,859,595, issued Aug. 22, 1989). In the absence of a rabbit-derived myeloma cell line, standard monoclonal antibody techniques are not useful. Thus, at present there is no reproducible method for generating rabbit monoclonal antibodies.
Some laboratories have looked at the production of heterohybrids. Antibody-secreting cells isolated from one species and fused with immortalizing cells from another species yield interspecies, or heterohybridomas. The term heterohybrid fusion is used herein interchangeably with xenogeneic fusions. Raybould, et al. disclose a method for producing rabbit-mouse hybridomas that secrete rabbit monoclonal antibodies (Raybould, et al., Science 240:1788-1790, 1988 and Raybould, et al., U.S. Pat. No. 4,977,081, issued Dec. 11, 1990). Rabbit monoclonal antibodies were produced by fusing mouse myeloma cells with rabbit splenocytes. Stable antibody production was only obtained when the fusion products were grown in the presence of rabbit serum. Raybould, et al. indicate in their conclusions that the use of rabbit serum is essential to rabbit monoclonal antibody production.
Rabbit monoclonal antibodies produced in the presence of rabbit serum is contaminated by endogenous rabbit antibodies present in the sera. Rabbit sera contains between 10-30 mg/ml of immunoglobulin G (IgG) as compared with microgram quantities of IgG produced by a murine hybridoma or heterohybridoma. Nonspecific rabbit immunoglobulin present in the monoclonal antibody preparation increases background reactivity and reduces the sensitivity of assays designed to detect antibody production from the fusion product. To prepare monoclonal antibodies for diagnostic assays, the monoclonal antibodies must be purified away from the nonspecific rabbit immunoglobulin present in the sera. These techniques are difficult, labor intensive and increase production costs.
Antibodies produced from interspecies hybridomas such as those methods disclosed by Raybould, et al. tend to be unstable. Because of the abnormal number of chromosomes, segregation does not always deliver identical sets of chromosomes to daughter cells and these chromosomes may be lost. Both the chromosomes containing the functional, rearranged immunoglobulin heavy-chain and light-chain genes and the chromosomes permitting drug resistance are needed to maintain cell replication and antibody production. As disclosed in the detailed description of the invention, (see Tables 2 and 3) the interspecies fusion of Raybould was unstable over time and failed to produce antibody.
The commercial use of a monoclonal antibody depends on the stability and quality of antibodies produced from a particular clone. In addition to stability and quality, the concentration of antibodies produced from the clone should be high enough, preferably greater than 25 .mu.g/ml, to obtain commercially useful quantities of antibodies. Usually the highest levels of antibody production are obtained when the antibodies are produced as ascites. Ascites production is the most cost effective and efficient way to grow large quantities of antibodies. Normally murine hybridomas are produced as ascites in a closely related murine host. Heterohybridomas secrete antibodies derived from an animal other than a mouse. An immunocompetent mouse will see the heterohybridomas as foreign or develop an immune response to the antibodies. Thus a method for producing rabbit monoclonal antibodies as ascites would represent a significant advance in the art.
While there is some evidence that rabbit heavy chain-murine light chain chimeric antibodies can be produced as ascites in nude mice, the use of mice to generate antibodies comprised of rabbit light and rabbit heavy chain is heretofore undisclosed (Dreher, et al., J. Immunology 130:442-447, 1983). Moreover, neither the chimeric clones nor those disclosed by Raybould were stable over time. Therefore useful quantities of antibodies for commercial diagnostic assays could not be produced. Ware, et al. demonstrated that rat X mouse hybridomas can be grown in severe combined immunodeficient (SCID) mice. (J. Immunological Methods 85:353-361, 1985, hereby incorporated by reference). Antibody production for xenogeneic fusions was previously limited to antibody production in tissue culture. The demonstration of high level heterohybridoma rabbit antibody production in SCID mice is heretofore undisclosed.
The ability to consistently produce rabbit monoclonal antibodies is undisclosed. Further, a method for reproducibly generating rabbit monoclonal antibodies in the absence of rabbit serum and a method for growing the antibody producing cells as an ascites tumor would be a significant advancement in the art. Rabbit monoclonal antibodies could thus be used to overcome some of the difficulties associated with generating antibodies to carbohydrate (Raybould, et al., supra).